Eukaryotic expression vectors are utilized for various biomedical applications including protein production, gene therapy and gene vaccination. A key barrier is that expression vectors undergo promoter inactivation (silencing) over time. This lack of sustained transgene expression negatively impacts the cost of cell culture production of recombinant proteins, and has limited the application of non-viral vector systems to short term applications. Here we propose to create enabling technology to improve the duration of expression from non-viral vectors in mammalian cells by developing novel eukaryotic expression vectors resistant to transgene silencing. These studies will utilize a eukaryotic expression vector backbone developed at NTC, containing a chimeric SV40-CMV promoter, which improves expression levels 10 fold over alternative CMV promoter vectors. We propose to further improve these vectors through evaluation of two methodologies to prevent transgene silencing. First, we hypothesize that transcription of the prokaryotic region will disrupt heterochromatin formation, and improve episomal or integrated cell line expression. We will transcribe the region of the plasmid that promotes heterochromatin formation using promoters integrated into the vector backbone. Second, we hypothesize that one or more structured regions within the replication origin form unusual structures that recruits heterochromatin and accounts for the silencing. High yield minimal origin constructs, that eliminate these putative heterochromatin inducing regions, will be constructed. The vectors will be tested in integrated and transiently transfected cell lines for improved performance. The overall goal of this feasibility study is to determine whether either strategy represents a significant advantage over alternate approaches to prevent transgene silencing, such as minicircle or matrix attached region (MAR) vectors. This technology, combined with the optimized NTC expression vectors, should enable NTC to create next generation expression systems for low cost production of cell culture derived recombinant proteins. In Phase II, NTC will make the vectors available for licensing, and will apply the technology to develop cell culture based methods for manufacture of glycoproteins such as FSH and biogeneric drugs. [unreadable] [unreadable] [unreadable]